Applied and Environmental Microbiology最新文献

5-Aminolevulinic acid (5-ALA) is a valuable precursor for pharmaceuticals and agriculture, but its microbial production is limited by tight coupling to essential heme biosynthesis. Here, we introduce a systematic, activity-graded tuning strategy for porphobilinogen synthase (PBGS, encoded by hemB) to decouple 5-ALA synthesis from heme metabolism in Corynebacterium glutamicum. Guided by structural and functional analyses, PBGS variants with progressively reduced activities were constructed to investigate the quantitative relationship between enzyme activity, cell growth, and 5-ALA accumulation. Controlled attenuation of PBGS activity maintained essential metabolism while markedly enhancing 5-ALA accumulation and minimizing porphyrin by-products. The engineered strain FA3 [hemB(D128E), hemA overexpression] achieved an optimal balance of growth and productivity. Metabolomic profiling confirmed that PBGS downregulation primarily suppressed porphyrin biosynthesis with minimal impact on central carbon metabolism. Subsequent metabolic and process optimizations, including gdhA and aceA deletion, dynamic rhtA expression, and cultivation control, further boosted production to 14.44 g/L 5-ALA in shake-flask culture, representing the highest shake-flask titer reported to our knowledge for C. glutamicum under similar conditions. This work provides the first systematic dissection of PBGS activity-dependent metabolic regulation and demonstrates that graded control of an essential enzyme enables rational metabolic decoupling, offering a broadly applicable framework for robust, high-yield microbial production of valuable compounds.IMPORTANCE5-Aminolevulinic acid (5-ALA) is an important precursor with pharmaceutical and agricultural applications, but microbial production is often constrained by its tight linkage to essential heme metabolism. Here, we systematically tuned porphobilinogen synthase activity to decouple 5-ALA accumulation from excessive porphyrin flux while maintaining cell growth. This strategy not only enabled the highest reported 5-ALA titer in Corynebacterium glutamicum but also highlights a broadly applicable framework for rationally engineering essential metabolic enzymes to achieve robust, high-yield microbial production of valuable compounds.

Methanogenic archaea that reside in the rumen of sheep, cattle, and other ruminants generate 16% of global emissions of methane, a potent greenhouse gas. The majority of rumen methanogens belong to species that display readily observable autofluorescence due to their intracellular co-factor, F₄₂₀. We developed a spectral flow cytometry method to directly quantify autofluorescent methanogens in the complex environment of the rumen. Rumen samples contain feed particles with natural autofluorescence signatures that overlapped those of F₄₂₀-containing methanogens. Spectral unmixing using natural autofluorescence signatures allowed us to distinguish methanogens from other autofluorescent particles and to quantify both cultured methanogens in buffer and native methanogens in rumen content samples over a concentration range from 4 × 10⁴ to 4 × 10⁷ cells/mL. The methanogen signal was absent in microbial cultures known to lack F₄₂₀ and in rumen content samples treated with sodium borohydride (NaBH₄), which reduces F₄₂₀ fluorescence. We showed a strong relationship between the number of autofluorescent methanogens in rumen content samples and methane yields in cattle and sheep treated with a methanogen inhibitor. We also assessed the impact of sample fixation on the spectral profiles of methanogen cells and showed that rumen samples stored at 4°C for up to 3 days remain suitable for enumeration. Our data thus demonstrate a new spectral flow cytometry method that can be used for rapid quantification of autofluorescent methanogens in rumen content samples.

Importance: Production of methane, a potent greenhouse gas, by methanogenic archaea in cattle, sheep, and other ruminants contributes around 16% of global methane emissions. Methane mitigation strategies are essential to respond effectively to the challenge of climate change, and many mitigations target rumen methanogens directly. In this study, we developed a method based on methanogen autofluorescence to identify and quantify methanogens within the complex rumen environment containing plant material that also fluoresces. The overlapping autofluorescence signals from methanogens and plant material can be resolved by applying spectral unmixing in spectral flow cytometry. This technique provides a rapid, practical approach for detecting and quantifying methanogens directly in rumen samples without the need for staining or additional fluorescent dyes or reagents. It provides a valuable tool to assess the impact of mitigation technologies. The method should also allow direct measurement of antibody binding to methanogens or determination of co-location of methanogens with other microbes.

Plant biomass degradation by fungal extracellular enzymes is tightly controlled at the transcriptional level, enabling fungi to respond to the complex and variable composition of plant cell wall. White-rot basidiomycetes are of special interest due to their unique ability to degrade all components of lignocellulose, yet information on their transcriptional regulators involved in this process is highly limited. In this study, we characterized DsAce3, a Zn(II)2Cys6 transcription factor from the white-rot fungus Dichomitus squalens, predicted to be orthologous to the cellulase activators Ace3 of the ascomycete fungus Trichoderma reesei and Roc1 of the basidiomycete fungus Schizophyllum commune. Disruption of DsAce3 suppressed extracellular cellulase production and significantly impaired growth on cellulose and cellobiose. Transcriptomic analyses also confirmed that DsAce3 is essential for activating not only cellulase- and xylanase-encoding genes but also sugar transporter-encoding genes, linking enzyme production to sugar sensing and uptake. DsAce3 shows a strong phylogenetic and functional conservation compared to its ascomycete counterpart in T. reesei, a rare feature in known regulators of lignocellulose degradation. However, differences in the response of DsAce3 to lactose indicate distinct substrate sensing or signaling mechanisms in basidiomycetes. This study describes the first functional characterization of an Ace3 ortholog in a strongly ligninolytic white-rot fungus and provides critical insights into transcriptional regulation in white-rot fungi, facilitating advances in fungal applications for biomass conversion.IMPORTANCEWhite-rot basidiomycete fungi are key organisms in natural carbon cycling due to their unique ability to break down all polymeric components of wood. Despite their ecological and biotechnological importance, little is known about how these fungi control the genes responsible for this process. In this study, we characterized a transcription factor, DsAce3, in the white-rot fungus Dichomitus squalens. We show that DsAce3 is essential for activating genes needed to degrade cellulose and import the sugars released. Notably, DsAce3 shares functional conservation with similar regulators in both ascomycete and basidiomycete fungi. Our findings offer new insights into how wood-decaying white-rot fungi regulate plant biomass degradation and provide a molecular foundation for improving fungal enzyme production in industrial and environmental applications.

Accumulating evidence suggests that exposure to low-hygiene environments (LHE) can alleviate chronic diseases by modulating the gut microbiota. Soil has been identified as a key factor in shaping microbial communities within the LHE. Furthermore, influenced by cultural customs and traditions, the practice of soil ingestion persists in many regions worldwide, serving both nutritional and medicinal purposes. However, whether soil confers efficacy comparable to that of LHE exposure or which intervention demonstrates better efficacy remains unexplored. This study compared the interventional effects and underlying mechanisms of sterile soil ingestion versus LHE exposure in dextran sulfate sodium (DSS)-induced chronic ulcerative colitis (UC) mice. Our results demonstrated that UC mice exhibited significant gut dysbiosis, characterized by reduced microbial diversity and disruption of microbial network structure. Both LHE exposure and soil ingestion markedly reshaped the gut microbial ecosystem, exhibiting highly similar regulatory patterns in microbial composition. LHE exposure increased fecal acetic acid concentrations, while soil ingestion elevated butyric acid levels. Moreover, both interventions effectively alleviated UC clinical symptoms, restored intestinal barrier integrity, and downregulated pro-inflammatory cytokine levels. In summary, sterile soil ingestion and LHE exposure exert comparable protective effects in UC mice. Given its operational simplicity and feasibility, sterile soil represents a promising translation and application. These findings support the potential of sterile soil as a practical therapeutic approach for alleviating UC-related gut ecological disruption in modern urban settings.IMPORTANCEUrbanization and excessive hygiene have reduced human exposure to soil and environmental microorganisms, contributing to the rising incidence of immune-related disorders such as inflammatory bowel disease. This study demonstrates that sterile soil ingestion and low-hygiene environment exposure comparably reshape the gut microbiota, enhance short-chain fatty acid production, and alleviate colitis in mice. These findings highlight sterile soil as a practical and controllable intervention to mimic the protective benefits observed in traditional, microbe-rich environments. Given the challenges of accessing low-hygiene settings in modern urban life, sterile soil represents a feasible therapeutic approach to alleviate gut dysbiosis and inflammation, with broad implications for microbiome-based therapeutics in industrialized societies.

Soybean is frequently nodulated by species from the Bradyrhizobium (BR) and/or Sinorhizobium (SR) genera. Several factors, such as soil pH, host genotype, geographic location, and other environmental variables, are reported to influence the preferential selection between BR and SR species within soybean root nodules. However, it remains unclear whether the age of the host plant at the time of inoculation affects preferential rhizobial selection. To investigate this, we inoculated soybean plants with different cell densities of BR and SR strains at three time points: at sowing (T₀), 2 weeks after germination (T₂), and 4 weeks after germination (T₄). We used 16S rRNA gene amplicon sequencing of root nodules and rhizosphere samples to assess the relative abundance of BR and SR in nodules and rhizosphere. We observed a clear shift in nodule occupancy that favored BR at the time of seed sowing (T₀) but increasingly favored SR when plants were inoculated at T₂ and T₄ stages. Specifically, at T₄, SR dominated in nodules across all treatments, representing 88%-99% of total sequences, regardless of applied inoculum ratio. In contrast, a similar number of sequences for both strains was detected in the rhizosphere at the time of the final harvest. These results highlight host age as an important ecological driver in legume-rhizobium interactions and suggest that inoculation time strongly influences microsymbiont selection. This information is important in understanding rhizobial competition and optimizing the timing of inoculation for soybeans.

Importance: Soybean is one of the world's most valuable crops and fulfills most of its nitrogen requirements by developing symbiotic associations with nitrogen-fixing rhizobia. This reduces the need for chemical fertilizers by converting atmospheric nitrogen into a plant-useable form of nitrogen. Multiple species from four rhizobial genera can nodulate soybean, and the plant's choice of rhizobial partner is reported to change depending on environmental conditions such as pH, host genotype, geographic location, and other environmental factors. This study explores how the age of the soybean plant affects its preference for two frequently reported beneficial rhizobial species (Bradyrhizobium diazoefficiens and Sinorhizobium fredii). By testing inoculation at different growth stages, we discovered that at early growth stages, plants favored Bradyrhizobium, while older plants increasingly selected SR for nodule formation. These findings highlight the level of complexity in plant-microbe interactions and could help optimize bioinoculant strategies for improving sustainability and crop yields.

Vibrio natriegens is an emerging bacterial platform for a range of biotechnological applications due to its rapid growth and ease of genetic manipulation. Whereas much has been learned about V. natriegens' aerobic physiology, comparatively little is known about its anaerobic fermentative physiology, despite its relevance to many industrial conditions. We compared the metabolic parameters of V. natriegens versus another biotechnologically relevant bacterium, Escherichia coli, under fermentative conditions. Both species excreted a similar array of fermentation products, but V. natriegens consumed less glucose and had a lower product titer. V. natriegens also exhibited rapid death, reaching extinction within 12 h after the growth phase, 3 days sooner than E. coli. Rapid V. natriegens death was avoided, and glucose consumption and product titers improved, by increasing the buffering capacity of the growth medium, indicating that V. natriegens is comparatively sensitive to its organic acid fermentation products. Aside from a minor role for RpoS in acid resistance, the V. natriegens genome lacks nearly all the acid resistance genes that have been characterized in E. coli and Vibrio cholerae. Our findings thus highlight an acid sensitivity that will need to be considered when designing fermentative applications of V. natriegens.

Importance: Bioprocessing, the biological conversion of renewable resources into value-added chemicals, is poised to meet an increasing demand for sustainable alternatives to petroleum-based products. Many examples of bioprocessing feature anoxic fermentations that naturally maximize product formation relative to growth of the microbial catalyst. Vibrio natriegens is a facultatively fermentative bacterium that has gained attention for bioprocessing due to its rapid growth rate and ease of genetic engineering. However, the fermentative properties of V. natriegens have not been compared to traditional bioprocessing workhorses like Escherichia coli. We revealed that V. natriegens is comparatively sensitive to its own acidic fermentation products, likely because V. natriegens lacks acid resistance mechanisms possessed by E. coli. Thus, fermentative applications must address this sensitivity either by buffering the fermentations, engineering resistance mechanisms, or bypassing the sensitivity by engineering V. natriegens to produce neutral products.

Although the metabolic pathways that allow the utilization of one-carbon compounds as sole sources of carbon and energy (methylotrophy) are well characterized, this understanding has been substantially refined and expanded in recent years. The paradigm-shifting discovery of the lanthanide-dependent methanol dehydrogenase, XoxF, established the biological relevance of rare-earth metals and revealed that methylotrophy required reassessment. We now know that XoxF is broadly distributed among bacteria and may actually constitute an ancestral form by which methylotrophy initially evolved, as well as the predominant form in which it now exists in nature. A new study published in Applied and Environmental Microbiology (C. R. Mineo, J. Jiang, and N. C. Martinez-Gomez, 91:e01304-25, 2025, https://doi.org/10.1128/aem.01304-25) extends this knowledge to characterize a heretofore undemonstrated methylotrophic pathway architecture among nitrogen-fixing plant symbionts of the Sinorhizobium and Bradyrhizobium genera. Their metabolic strategy proceeds via XoxF, complete oxidation to carbon dioxide, and the Calvin-Benson-Bassham cycle to assimilate the oxidized carbon. The authors designate this the "XoxF-CBB pathway," which appears to be well-conserved across these groups of bacteria. Their streamlined pathway represents a unique connection between autotrophy and methylotrophy that, when paired with XoxF, could constitute an underappreciated, but prevalent, variation on methylotrophy. The study highlights the need to remain open-minded about methylotrophic pathway configurations in bacteria, as well as informing the ways in which we should consider seeking to isolate novel methylotrophs. Finally, the pathway's presence in nodule-forming bacteria raises new questions about how methylotrophy shapes their physiology in both free-living soil conditions and plant-symbiotic associations.

Fish protein hydrolysates hold great promise as nutraceuticals, yet their application as food ingredients or nutraceuticals is currently limited by their fish-like odor. This odor is mainly due to the presence of trimethylamine (TMA), a volatile biogenic amine resulting from the breakdown of naturally occurring trimethylamine-N-oxide (TMAO) in marine fish. The bacterial trimethylamine monooxygenase mFMO can oxidize TMA into TMAO using molecular oxygen and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). We have established an enzyme cascade that takes advantage of glucose dehydrogenase to recycle NADPH from NADP⁺, significantly decreasing the cost of the reaction and paving the way for using the enzyme system in fish protein hydrolysates targeted for human consumption. We demonstrate that the dual enzyme system works in an industrially relevant substrate. Salmon protein hydrolysate treated with an mFMO/glucose dehydrogenase cocktail showed a 75% reduction in TMA. A trained sensory panel perceived an improved odor across several parameters, including a reduction in the characteristic TMA smell.IMPORTANCEMarine by-products are a valuable source of high-quality peptide ingredients; however, their application in the food market is limited by the unpleasant fishy odor caused by trimethylamine (TMA). An enzyme that oxidizes TMA to the odor-free trimethylamine-N-oxide (TMAO) in salmon protein hydrolysates is known, but it requires excessive amounts of NADPH, an expensive cofactor. Here, we describe a cofactor regeneration system that allows using less cofactor in the enzyme-driven TMA removal process. This dual enzyme system removed 75% of TMA from a salmon protein hydrolysate, resulting in a significantly reduced fishy odor as confirmed by a trained sensory panel compared to the untreated control. This enzyme cascade is an important step toward making targeted TMA removal economically feasible for marine biomass valorization.

Groundwater acidification co-occurring with nitrate pollution is a common, global environmental health hazard. Denitrifying bacteria have been leveraged for the in situ removal of nitrate in groundwater. However, co-existing stressors-such as low pH-reduce the efficacy of biological removal processes. Castellaniella sp. str. MT123 is a complete denitrifier that was isolated from acidic, nitrate-contaminated groundwater. The strain grows robustly by nitrate respiration at pH < 6.0, completely reducing nitrate to dinitrogen gas. Genomic analyses of MT123 revealed few previously characterized acid tolerance genes. Thus, we utilized a combination of proteomics, metabolomics, and competitive mutant fitness to characterize the genetic mechanisms of MT123 acclimation to growth under mildly acidic conditions. We found that glutamate accumulation is critical in the acid acclimation of MT123, possibly through consumption of intracellular protons via glutamate decarboxylation to GABA. This is despite the fact that MT123 lacks the canonical glutamate decarboxylase-glutamate/GABA antiporter system implicated in acid tolerance in other bacteria. In contrast, branched-chain amino acid (BCAA) accumulation was detrimental to cell growth at lower pHs, possibly through indirect mechanisms impacting the cellular glutamate pool. Genetic analysis previously linked MT123 to a population of Castellaniella that bloomed-concurrent to nitrate removal-during a biostimulation effort to reduce groundwater nitrate concentrations at MT123's location of origin. Thus, our analyses provide novel insight into mechanisms of acclimation to acidic conditions in a strain with significant potential for nitrate bioremediation.IMPORTANCENitrate pollution in groundwater is a major threat to both environmental and human health. This nitrate pollution can come from a variety of sources, including farm fertilizers, sewage, animal waste, septic systems, and industrial discharge. Bacteria known as "denitrifiers" can convert this nitrate into harmless nitrogen gas, a process known as "denitrification." Denitrifiers can be used to clean up nitrate-contaminated groundwater. However, their ability to do this can be disrupted by changing environmental conditions. For example, groundwater that is polluted with nitrate is often acidic. Acidic conditions make it challenging for denitrifiers to survive, which results in less conversion of nitrate to nitrogen gas. In this study, we investigated how one denitrifying bacterium-originating from acidic, nitrate-contaminated groundwater-can cope with acidic conditions.